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. 2021 Oct 13;21:1101. doi: 10.1186/s12885-021-08841-1

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 6 — Proteasome activity was required for MTX-induced apoptosis. A. Proteasomes activation assay. LNCaP and 22RV1 cells were treated with vehicle, MTX (1 μM) for 24 h. Proteasome activation of LNCaP (up) and 22RV1(down) treated with indicated concentrations were indicated as hydrolysis rate succinyl-Leu-Leu-Val-Tyr-amc (Suc-LLVY-amc). B-C. LNCaP cells were treated with vehicle, BZM (200 nM), MTX (1 μM) alone and combination (BZM, 100 nM; MTX, 0.5 μM) for 24 h. B. Representative flow cytometry plots. C. Quantitative data of apoptosis analysis. Early apoptotic was defined as Annexin positive population, later apoptotic as Annexin/PI positive. D. Cells proliferation was determined by CCK-8. Data are presented from three independent experiments. All data are presented as mean ± s.e.m. The asterisk indicates a significant difference. (One-way ANOVA, *p < 0.05; **p < 0.01)